Desensitization of G protein-coupled receptors such as the beta2- adrenergic receptor and the visual receptor rhodopsin occurs concomitantly with receptor phosphorylation. Recent evidence supports the involvement of a stimulus-dependent receptor phosphorylation in the desensitization of these receptors. This phosphorylation, mediated by the enzymes rhodopsin kinase and beta-adrenergic receptor kinase (betaARK), then promotes the binding of a second protein, arrestin or beta-arrestin, with the phosphorylated receptor. Arrestin/beta-arrestin binding to the receptor appears to be largely responsible for decreasing receptor/G protein coupling and thereby mediating desensitization. although both retinal arrestin and beta-arrestin have been cloned, little is presently known about the molecular mechanisms, specificity, and structural features involved in arrestin/receptor interaction. The major objectives of this proposal are to elucidate the molecular mechanisms involved in arrestin/receptor interaction and the role that arrestins play in mediating desensitization of G protein-coupled receptors. Initial studies have focused on identifying additional members of the arrestin gene family. To date, we have cloned one new arrestin, termed arrestin3, and identified polypeptide variants of both arrestin3 and beta-arrestin. Low stringency hybridization and the polymerase chain reaction will be used to isolate additional arrestins. In vitro translation and the insect cell/baculovirus system have been used to express and characterize the different mammalian arrestins. These expression systems, in addition to the production of mammalian cell lines stably transfected with different arrestins, should enable a detailed characterization of arrestin function both in intact cells and in vitro. Receptor desensitization will be studied using pharmacological and arrestin translocation assays in cell lines that express endogenous beta-arrestin and/or arrestin3 (determined by immunoblotting and immunofluorescence with specific antibodies) or in cell lines in which expression has been decreased using antisense strategies. The production of various mutant and chimeric arrestin/beta-arrestin molecules has helped to identify the structural features of arrestins which are important for function. Experiments studying arrestin binding to mutant receptors or binding in the presence of receptor peptides will also help to localize receptor domains important for arrestin binding. The specificity of interaction of the arrestins with distinct receptor classes will be assessed utilizing intact cell assays, co-expression studies and in vitro reconstitution assays with native and recombinant components. In addition, we will test the possibility that arrestins also interact with phospholipase C. Finally, the potential role of direct phosphorylation of the arrestins (and its functional consequence) will be characterized using intact cell phosphorylation, in vitro phosphorylation and mutagenesis.